Bacteriocinogenic Lactobacillus plantarum ST16Pa isolated from papaya (Carica papaya) — From isolation to application: Characterization of a bacteriocin

Strain ST16PA, isolated from papaya was identified as Lactobacillus plantarum based on biochemical tests, PCR with species-specific primers and 16S rDNA sequencing. L. plantarum ST16PA produces a 6.5 kDa bacteriocin, active against different species from genera Enterobacter, Enterococcus, Lactobacillus, Pseudomonas, Streptococcus and Staphylococcus and different serotypes of Listeria spp. The peptide is inactivated by proteolytic enzymes, but not when treated with ??-amylase, catalase, lipase, Triton X-100, SDS, Tween 20, Tween 80, urea, NaCl and EDTA. However, presence of 1% Triton X-114 deactivates the bacteriocin. No change in activity was recorded after 2 h at pH values between 2.0 and 12.0, and after treatment at 100 °C for 120 min or 121 °C for 20 min. The mode of activity against Lactobacillus sakei ATCC 15521, Enterococcus faecalis ATCC 19443 and Listeria innocua 2030C was bactericidal, resulting in cell lysis and enzyme-leakage. No significant differences in cell growth and bacteriocin production were observed when strain ST16Pa was cultured in MRS broth at 26 °C and 30 °C for 24 h (25 600 AU/ml). However, even though strain ST16PA grows well in MRS broth at 15 °C and 37 °C, a reduction of bacteriocin production was observed (400 AU/ml and 1600 AU/ml, respectively). In addition, effect of MRS medium components, different initial pH and additions of glycerol or vitamins to the media on bacteriocin ST16Pa production was studied.Peptide ST16PA adsorbs (400 AU/ml) to producer cells. However, bacteriocin ST16Pa was adsorbed at 50% to cells of L. innocua 2030C and at 75% to L. sakei ATCC 15521 and E. faecalis ATCC 19433 when experiments were conducted at 30 °C and pH 6.5. Adsorption of bacteriocin ST16Pa to target cells at different temperatures, pH and in presence of potassium sorbate, sodium nitrate, sodium chloride, ascorbic acid, Tween 80 and Tween 20 were also studied. To the best of our knowledge, this is the first report on detection of L. plantarum in papaya.     

Biochemical,antimicrobial and molecular characterization of a noncytotoxic bacteriocin produced by Lactobacillus plantarum ST71KS

Lactobacillus (Lb.) plantarum ST71KS was isolated from homemade goat feta cheese and identified using biochemical and molecular biology techniques. As shown by Tricine-SDS-PAGE, this lactic acid bacterium produces a bacteriocin (ST71KS) with an estimated molecular weight of 5.0 kDa. Bacteriocin ST71KS was not affected by the presence of α-amylase, catalase and remained stable in a wide range of pH and after treatment with Triton X-100, Triton X-114, Tween 20, Tween 80, NaCl, SDS, urea and EDTA. This bacteriocin also remained active after being heated at 100 °C for 2 h and even after 20 min at 121 °C; however, it was inactivated by proteolitic enzymes. Production of bacteriocin ST71KS reached 6400 AU/mL during stationary growth phase of Lb. plantarum cultivated in MRS at 30 °C and 37 °C. Bacteriocin ST71KS displayed a bactericidal effect against Listeria monocytogenes strains 603 and 607 and did not adsorb to the producer cells. Lb. plantarum ST71KS harbors two bacteriocin genes with homology to plantaricin S and pediocin PA-1. These characteristics indicate that bacteriocin ST71KS is a class IIa bacteriocin. The peptide presented no toxic effect when tested in vitro with kidney Vero cells, indicating safe technological application to control L. monocytogenes in foods.     

Artocarpus integer leaf protease: Purification and characterisation

The presence of a protease in Artocarpus integer leaves, which are traditionally used as a meat tenderiser, was verified by the presence of a band at 69 kDa, using caseinolytic zymography. Purification by temperature phase partitioning with Triton X-114, ammonium sulphate precipitation and gel filtration chromatography yielded a preparation with a 12-fold increase in enzyme purity and a final specific activity of 76.67 U/mg. The cysteinic nature of this enzyme was confirmed through inhibition of enzyme activity by E-64 and iodoacetamide and enhancement of activity by cysteine and 2-mercaptoethanol. The protease retained 70% of its activity over a broad pH range (pH 6–12), with optimal activity recorded at pH 10 and 40 °C. The enzyme was stable at temperatures up to 70 °C, with 80% of its activity intact. Addition of 5 mM Ca²⁺ stimulated enzyme activity and a kinetic study of the enzyme yielded K_m and V_max values of 0.304 mg/mL and 0.735 mg/mL/min, respectively.     

Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: Its purification and characterization

Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5 kDa according to SDS–PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40 °C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe²⁺ > Hg²⁺ > Mn²⁺ > K⁺ > Mg²⁺ > Li⁺ > Cu²⁺). Trypsin activity decreased continuously as NaCl concentration increased (0–30%). K_m and k_cat values were 0.13 mM and 1.46 s⁻¹, respectively. Results suggest the enzyme have a potential application where room processing temperatures (25–35 °C) or high salt (30%) concentration are needed, such as in fish sauce production.     

Trace element content of Boletus tomentipes mushroom collected from Yunnan,China

The contents of As, Cd, Cr, Cu, Fe, Hg, Mg, Mn, Ni, Pb, and Zn in eleven fruiting bodies of Boletus tomentipes were determined. The results showed the values of the studied elements decreased in the order: Mg (208–279 mg kg⁻¹) > Fe (106–137 mg kg⁻¹) > Mn (29.5–46.8. mg kg⁻¹) > Zn (18.7–23.1 mg kg⁻¹), > Cu (11.4–15.8 mg kg⁻¹) > Cr (3.36–4.78 mg kg⁻¹) > Pb (1.38–3.88 mg kg⁻¹) > Ni (1.68–3.01 mg kg⁻¹) > Cd (0.16–0.32 mg kg⁻¹) > As (0.10–0.24 mg kg⁻¹) > Hg (<0.06 mg kg⁻¹).     

Amaranthus hypochondriacus and Amaranthus caudatus germplasm: Characteristics of plants,grain and flours

In the present study, the plant, grain and flour characteristics of 48 Amaranthus hypochondriacus and 11 Amaranthus caudatus lines are reported. A. hypochondriacus grains had a higher thousand kernel weight (0.62–0.88 g), a creamish yellow colour (L∗ = 61.38–68.29, b∗ = 5.26–6.8 and a∗ = 19.71–23.84), whereas A. caudatus grains had a lower thousand kernel weight (0.46–0.70 g) and a reddish-brown colour (L∗ = 47.1–51.2, b∗ = 11.82–14.02 and a∗ = 7.72–13.29). The A. caudatus lines had a higher protein content, fat content and tendency for retrogradation, and lower α-amylase activity as compared to A. hypochondriacus lines. The Amaranthus lines with higher a∗-values and lower thousand kernel weight, L∗- and b∗-values, had a higher fat, crude fibre and protein content. The correlation of fat and crude fibre content with the thousand kernel weight, L∗-, a∗- and b∗-values, was significant. The A. hypochondriacus lines showed higher pasting temperature, and lower peak viscosity, breakdown and setback, as compared to the A. caudatus lines. The result reflected that the lines with higher α-amylase activity showed a lower peak viscosity, breakdown, final viscosity and gel hardness, and a higher pasting temperature.     

Characterisation of acid protease expressed from Aspergillus oryzae MTCC 5341

Aspergillus oryzae (MTCC 5341) has the largest expanse of hydrolytic genes, that includes 135 protease genes coding for alkaline, acid as well as neutral proteases. This study reports the purification and characterisation of an acid protease obtained from A. oryzae MTCC 5341. A. oryzae MTCC 5341 produces one of the highest reported acid protease activities reported so far (8.3 × 10⁵ U/g dry bran). The extracellular acid protease (47 kDa) was found to be active in the pH range 3.0–4.0 and stable in the pH range 2.5–6.5. Optimum temperature for activity was 55 °C. The protease was purified 17–fold with a yield of 29%. The enzyme was characterised to be an aspartate protease by inhibition studies, using pepstatin and its ability to activate trypsinogen. The enzyme cleaved the B-chain of insulin at L–V and Y–T residues.     

Purification and characterization of transglutaminase from a newly isolated Streptomyces hygroscopicus

Transglutaminase (TGase, EC 2.3.2.13) from a Streptomyces hygroscopicus strain isolated from soil was purified from culture broth by ethanol precipitation, followed by successive chromatographies on CM-cellulose and Sephadex G-75 columns with a yield and purification-fold of 21.1% and 30%, respectively. The enzyme’s molecular weight was estimated as 38,000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified microbial transglutaminase (MTG) exhibited optimum activity at 37–45 °C and in a range of pH 6.0–7.0 for hydroxamate formation from N-carboxybenzoyl-l-glutaminyl-glycine and hydroxylamine. The enzyme was not stable above 50 °C and was stable within a pH range of 5.0–8.0 at lower temperature. The MTG was not inhibited by Ca²⁺ and ethylenediaminetetraacetic acid, suggesting it was calcium-independent. Purified MTG was strongly inactivated by 5,5′-dithiobis (2-nitrobenzoic acid), Cu²⁺, Zn²⁺, Pb²⁺, and Hg²⁺, suggesting that this enzyme could possess a thiol group at the active site. The MTG stability was strongly affected by ethanol concentration. The enzyme activity was slightly elevated at a lower concentration of ethanol at 25 °C.     

Kinetic modeling of spice extraction from S. aromaticum and C. cassia

A mathematical model was developed for the extracts obtained from Syzygium aromaticum and Cinnamomum cassia with different particle size, solvent–solid ratios on extraction yield. Different particle sizes in the range of 2.8 mm to ?0.5 mm were employed and maximum extraction efficiency was achieved with particles of size ?0.5 mm. Among the solvent–solid ratios (20:1, 30:1, 40:1 and 50:1) ratio of 50:1 showed higher extraction yield. In the extraction kinetics, higher effective diffusivity value of 36.01 × 10⁻¹⁰ m²/s for S. aromaticum and 26.78 × 10⁻¹⁰ m²/s for C. cassia were achieved. Antioxidant values were determined and extracts prepared from ethanol showed higher scavenging activities for S. aromaticum and C. cassia as 78% and 85% respectively. Maximum phenolic content of 1.6 and 12.4 mg GAE/g of sample were achieved for S. aromaticum and C. cassia by hexane and water respectively.     

Use of Fourier transform infrared spectroscopy for rapid comparative analysis of Bacillus and Micrococcus isolates

The identification of foodborne microorganisms and their endospores in food products are important for food safety. The present work compares Bacillus (Bacillus licheniformis, Bacillus circulans and Bacillus subtilis) and Micrococcus (Micrococcus luteus) species with Fourier transform infrared (FTIR) spectroscopy. Our results show that there are several characteristic peaks belonging to both the Micrococcus and Bacillus species which can be used for the identification of these foodborne bacteria and their endospores. For Micrococcus species, a new band was observed at 1338 cm⁻¹ which may be due to acetate oxidation via the carboxylic acid cycle. The bands at 1313 cm⁻¹ and 1256 cm⁻¹ can be explained by an exopolymer formation and the other bands at 1074 cm⁻¹ and 550 cm⁻¹, may be due to the glycogen-like storage material in Micrococcus spp. There are also characteristic peaks at 993 cm⁻¹ and 801 cm⁻¹ for these bacterial species. Different Bacillus species also showed characteristic peaks at 1000–500 cm⁻¹ region. Dipicolinic acid (DPA) bands at ∼728 cm⁻¹ and ∼703 cm⁻¹ seen only in B. circulans were the marker of an endospore formation.     

Partial purification and characterisation of endoglucanase from an edible mushroom, Lepista flaccida

A crude extract was prepared from the fruiting body of Lepista flaccida, an edible mushroom and endoglucanase activity of the extract was increased 14-fold with ammonium sulphate precipitation. Maximum enzyme activity was seen at pH 4.0 and 50 °C when carboxymethylcellulose was used as a substrate. K_0.5 and V_max values of the partially purified endoglucanase were 7.7 mg/ml and 25 ± 0.9 U/mg protein, respectively. The enzyme was quite stable over a broad range of pH (2.0–9.0) at 4 °C. When it was incubated at temperatures between 20 °C and 60 °C for 12 h, it conserved much of its original activity (over 40%). The activity of the enzyme increased by 234 ± 3.6% in the presence of 1 mM Mn²⁺. The endoglucanase was inhibited by EDTA, PMSF, β-ME and DDT. In conclusion, pH and thermal stability of the L. flaccida endoglucanase could make it useful for industrial purposes.     

Water adsorption isotherms of microcapsules with hydrolyzed pinhão (Araucaria angustifolia seeds) starch as wall material

Water adsorption isotherms of microcapsules that use pinhão starch as the wall material and β-carotene as the core material were evaluated. Pinhão was chosen as the raw material because it is primarily composed of starch, and in the literature there are few technological aspects with regard to this polymer. Moreover, this wall material has been previously studied and demonstrated good encapsulation properties. Moisture adsorption isotherms were determined at 10, 20 and 30 °C using the gravimetric method. The Peleg model was used to fit the experimental data. The differential enthalpy and entropy of sorption were calculated using moisture sorption data; both decreased as moisture content increased. The enthalpy–entropy compensation theory was applied to adsorption isotherms and the isokinetic temperature was calculated by plotting the differential enthalpy as a function of differential entropy. We found that the adsorption process was enthalpy-controlled, the T_β (352.61 K) was higher than the T_hm (292.77 K), and the process was spontaneous (ΔG = −470.17 J mol⁻¹).     

Chemical composition,antioxidant and antibacterial activities of the essential oils isolated from Tunisian Thymus capitatus Hoff. et Link.

The chemical composition, antioxidant and antibacterial activities of essential oils isolated by hydrodistillation from the aerial parts of Tunisian Thymus capitatus Hoff. et Link. during the different phases of the plant development, and from different locations, were evaluated. The chemical composition was analyzed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The main components of the essential oils were carvacrol (62–83%), p-cymene (5–17%), γ-terpinene (2–14%) and β-caryophyllene (1–4%). The antioxidant activity of the oils (100–1000 mg l⁻¹) was assessed by measurement of metal chelating activity, the reductive potential, the free radical scavenging (DPPH) and by the TBARS assay. The antioxidant activity was compared with that of synthetic antioxidants: butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Both the essential oils and BHA and BHT showed no metal chelating activity. Although with the other methodologies, there was a general increase in the antioxidant activity, with increasing oil concentration, maxima being obtained in the range of 500 and 1000 mg l⁻¹ for flowering and post-flowering phase oils. Major differences were obtained according to the methodology of antioxidant capacity evaluation. Antibacterial ability of Th. capitatus essential oils was tested by disc agar diffusion against Bacillus cereus, Salmonella sp., Listeria innocua, four different strains of Staphylococus aureus (C15, ATCC25923, CFSA-2) and a multi-resistant form of S. aureus (MRSA-2). Antibacterial properties were compared to synthetic antibiotics. Higher antibacterial activity was observed with the flowering and the post-flowering phase essential oils.     

Characterisation of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L.)

Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification – by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose – was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0–11.0 and an optimal temperature of approximately 55–60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis–Menten constant (K_m) and catalytic constant (K_cat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s⁻¹, respectively. The catalytic efficiency (K_cat/K_m) was 238 s⁻¹ mM⁻¹.     

Identification,quantification and antioxidant activity of acylated flavonol glycosides from sea buckthorn (Hippophae rhamnoides ssp. sinensis)

A novel acylated flavonol glycoside: isorhamnetin (3-O-[(6-O-E-sinapoyl)-β-d-glucopyranosyl-(1 → 2)]-β-d-glucopyranosyl-7-O-α-l-rhamnopyranoside) (1), together with two known acylated flavonol glycosides: quercetin (3-O-[(6-O-E-sinapoyl)-β-d-glucopyranosyl-(1 → 2)]-β-d-glucopyranosyl-7-O-α-l-rhamnopyranoside) (2) and kaempferol (3-O-[(6-O-E-sinapoyl)-β-d-glucopyranosyl-(1 → 2)]-β-d-glucopyranosyl-7-O-α-l-rhamnopyranoside) (3) were isolated from the n-butanol fraction of sea buckthorn (Hippophae rhamnoides ssp. sinensis) berries for the first time by chromatographic methods, and their structures were elucidated using UV, MS, ¹H and ¹³C NMR, and 2D NMR. Compounds 1–3 showed good scavenging activities, with respective IC₅₀ values of 8.91, 4.26 and 30.90 μM toward the 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical; respective Trolox equivalent antioxidant capacities of 2.89, 4.04 and 2.44 μM μM⁻¹ toward 2,2′-azino-bis-3-ethyl-benzothiazoline-6-sulphonate (ABTS) radical. The quantitative analysis of the isolated acylated flavonol glycosides was performed by HPLC–DAD method. The contents of compounds 1–3 were in the range of 12.2–31.4, 4.0–25.3, 7.5–59.7 mg/100 g dried berries and 9.1–34.5, 75.1–182.1, 29.2–113.4 mg/100 g dried leaves, respectively.     

Effect of low voltage electrical stimulation on biochemical and quality characteristics of Longissimus thoracis muscle from one-humped Camel (Camelus dromedaries)

The effects of electrical stimulation (90 V) 20 min post mortem on meat quality and muscle fibre types of four age group camels (1–3, 4–6, 7–9, 10–12 years) camels were assessed. Quality of the Longissimus thoracis at 1 and 7 days post mortem ageing was evaluated using shear force, pH, sarcomere length, myofibrillar fragmentation index, expressed juice, cooking loss and L^∗, a^∗, b^∗ colour values. Age of camel and electrical stimulation had a significant effect on meat quality of L. thoracis. Electrical stimulation resulted in a significantly (P < 0.05) more rapid pH fall in muscle during the first 24 h after slaughter. Muscles from electrically-stimulated carcasses had significantly (P < 0.05) lower pH values, longer sarcomeres, lower shear force value, higher expressed juice and myofibrillar fragmentation index than those from non-stimulated ones. Electrically-stimulated meat was significantly (P < 0.05) lighter in colour than non-stimulated based on L^∗ value. Muscles of 1–3 year camels had a significantly (P < 0.05) lower shear force value, and pH, but longer sarcomere, and higher myofibrillar fragmentation index, expressed juice, and lightness colour (L^∗) than those of the 10–12 years camels. The proportions of Type I, Type IIA and Type IIB were 25.0, 41.1 and 33.6%, respectively were found in camel meat. Muscle samples from 1–3 year camels had significantly (P < 0.05) higher Type I and lower Type IIB fibres compared to those from 10–12 year camel samples. These results indicated that age and ES had a significant effect on camel meat quality.     

Antioxidant and antibacterial activities of exopolysaccharides from Bifidobacterium bifidum WBIN03 and Lactobacillus plantarum R315

The objective of this study was to investigate the antioxidant and antibacterial activities of exopolysaccharide (EPS) from Bifidobacterium bifidum WBIN03 (B-EPS) and Lactobacillus plantarum R315 (L-EPS). The 1,1-diphenyl-2-picrylhydrazyl (DPPH)-radical scavenging, hydroxyl radical-scavenging, and superoxide radical-scavenging abilities were measured to evaluate antioxidant activity. Inhibition of erythrocyte hemolysis and lipid peroxidation was also measured. Both B-EPS and L-EPS had strong scavenging ability against DPPH and superoxide radicals at high concentration. The inhibitory effect of B-EPS on erythrocyte hemolysis was stronger than that of L-EPS in a concentration range from 0.30 to 1.00 mg/mL, whereas the hydroxyl scavenging ability of L-EPS (39.15 ± 0.58%) was significantly higher than that of 0.15 mg/mL ascorbic acid (24.33 ± 1.17%) and B-EPS (17.89 ± 3.30%) at 0.10 mg/mL. The inhibition of lipid peroxidation of 0.50 mg/mL B-EPS and L-EPS was 13.48 ± 1.74% and 12.43 ± 0.51%, respectively, values lower than that of ascorbic acid at the same concentration (23.20 ± 1.41%). Furthermore, all these abilities were enhanced in a concentration-dependent manner. Agar diffusion assay showed that both EPS exhibited antibacterial activities against tested pathogens such as Cronobacter sakazakii, Escherichia coli, Listeria monocytogenes, Staphyloccocus aureus, Candida albicans, Bacillus cereus, Salmonella typhimurium, and Shigella sonnei at 300 μg/mL. In conclusion, both EPS have antimicrobial and antioxidant activities and could have applications in the food industry.     

Effects of Kudoa spores,endogenous protease activity and frozen storage on cooked texture of minced Pacific hake (Merluccius productus)

The effects of Kudoa infection of Pacific hake (Merluccius productus) on endogenous protease activity and on cooked mince texture were investigated. Texture was significantly (p < 0.05) negatively correlated with spore counts as well as protease activity. Soft texture (maximum force <150 g) was observed in fish with 10⁴–10⁶Kudoa thyrsites spores g⁻¹ mince, compared to 10⁵–10⁸K. paniformis spores g⁻¹ mince, suggesting that especially for fish having lower infection levels, K. thyrsites may have a greater impact than K. paniformis on Pacific hake quality. Pre-incubation for 15 min at 52 °C prior to cooking resulted in softer texture in some samples due to endogenous proteolytic action. This pre-incubation effect was not consistently observed in fish held 6 months or longer at −25 °C or after freeze-thaw cycling, which may be explained by an opposing toughening effect attributed to protein denaturation and aggregation during prolonged or abusive frozen storage.     

Profiling of in vitro neurobiological effects and phenolic acids of selected endemic Salvia species

The ethyl acetate and methanol extracts from 16 Salvia L. species were screened for their inhibitory activity against acetylcholinesterase, butyrylcholinesterase, lipoxygenase, and tyrosinase; the enzymes linked to neurodegeneration. Their antioxidant activity was also tested using DPPH radical scavenging, metal-chelation, and ferric-reducing antioxidant power (FRAP) assays. Total flavonoid content of the extracts was determined by AlCl₃ reagent, while HPLC technique was applied for analysis of various phenolic acids in the extracts. The extracts exerted weak cholinesterase and tyrosinase inhibition, and remarkable inhibition against lipoxygenase (13.07 ± 2.73-74.21 ± 5.61%) at 100 μg ml⁻¹. The methanol extracts showed higher antioxidant activity in DPPH radical scavenging and FRAP assays. The extracts were analyzed for their gallic, protocateuchic, p-hydroxy-benzoic, vanillic, caffeic, chlorogenic, syringic, o- and p-coumaric, ferulic, rosmarinic, and tr-cinnamic acid contents and the methanol extract of Salvia ekimiana (153.50 mg 100 g⁻¹) was revealed to be the richest in terms of rosmarinic acid.